The gene of an archaeal a-l-fucosidase is expressed by translational frameshifting

Cobucci-Ponzano, Beatrice; Conte, Fiorella; Benelli, Dario; Londei, Paola; Flagiello, Angela; Monti, Maria; Pucci, Piero; Rossi, Mosè; Moracci, Marco

The experimental data reported above indicate that the predicted slippery heptanucleotide in the region of overlap between the ORFs SSO11867 and SSO3060 of the wildtype gene fucA1 could regulate in cis the frameshifting events observed in E.coli. To test this hypothesis, we mutated the sequence A-AAA-AAT into A-AAG-AAT and C-AAG-AAC (mutations are underlined) obtaining the fucA1 single mutant ( fucA1 sm ) and triple mutant ( fucA1 tm ) genes, respectively. It is worth noting that the mutations disrupt the slippery sequence, but they maintain the À1 frameshift between the two ORFs (Table 1) . Surprisingly, the expression of fucA1 sm in E.coli produced a full-length polypeptide that, after purification by affinity chromatography and removal of the GST protein, showed the same electrophoretic migration of Ssa-fuc and Ssa-fuc B ( Figure 4A ). This protein was then characterized by mass spectrometry analyses following in situ tryptic digestion. Interestingly, the MALDI spectra revealed the presence of a single peptide encompassing the overlapping region between the two ORFs with a mass value of 1259.7 Da (peptide C; Figure 4B ). The sequence of peptide C, determined from the fragmentation spectra obtained by LCMSMS analysis, was Glu-Phe-Gly-Pro-Val-Thr-Asp-Phe-Gly-Tyr-Lys ( Figure 4C ). Remarkably, apart from the Glu residue, this sequence is identical to that of peptide B produced from fucA1, indicating that in the mutant gene fucA1 sm only one of the two frameshifting events observed in the wild-type fucA1 gene had occurred. The presence of a Glu instead of Lys was not unexpected. The mutation A-AAA-AAT!A-AAG-AAT in fucA1 sm was conservative in the zero frame of the ORF SSO11867 (AAA!AAG, both encoding Lys), but it produced the mutation AAA!GAA (Lys!Glu) in the À1 frame of the ORF SSO3060.